NeuroMab™ Anti-CXCL12 BBB Shuttle Antibody(NRZP-1022-ZP3789)

Figure 1. Anti-CXCL12 antibody inhibits cancer cell migration.

Transwell migration of fluorescently labeled TOV-21G cells into CXCL12 was quantified using fluorescence scanning. 114_3H1 (labeled 114_3H01) and 113_1H12 IgG titrated from 0.39-500 nM were mixed with 80 ng/ml (10 nM) human CXCL12 in the lower chamber to test the effect of these antibodies on CXCL12-induced migration. Anti-lysozyme antibody (500 nM) was used as an isotype control. All error bars represent standard deviation.

Figure 2. Anti-CXCL12 antibody inhibits angiogenesis.

Human umbilical vein endothelial cells (HUVEC) were seeded onto fibroblasts grown for 6 days on gelatin-coated chamber slides. These two cell types were co-cultured for 7 days in medium containing VEGF and lead anti-CXCL12 antibodies 114_3H1 and 113_1H12. IgG bound to lysozyme (nonspecific IgG) was used as an isotype control for the assay (Panel A). After 7 days of co-culture, cells were stained for platelet/endothelial adhesion molecule-1 (PECAM-1, an angiogenic marker) to visualize tubule formation and branching by light microscopy. Use AngioSys image analysis software to calculate the total number of tubules and the number of branch connections.

Figure 3 Anti-CXCL12 antibody inhibits angiogenesis.

Human umbilical vein endothelial cells (HUVEC) were seeded onto fibroblasts grown for 6 days on gelatin-coated chamber slides. These two cell types were co-cultured for 7 days in medium containing VEGF and lead anti-CXCL12 antibodies 114_3H1 and 113_1H12. IgG bound to lysozyme (nonspecific IgG) was used as an isotype control for the assay (Panel A). After 7 days of co-culture, cells were stained for platelet/endothelial adhesion molecule-1 (PECAM-1, an angiogenic marker) to visualize tubule formation and branching by light microscopy. Use AngioSys image analysis software to calculate the total number of tubules, total tubule length (Figure 6B).

Figure 4A shows migration trajectories of the results of experiments to determine the effectiveness of antibodies (113_1H12 (hAB113) in the human IgG2 form, 113_1H12 (mAB113) in the chimeric mouse IgG2a form, and 114_3H1 (mAB114) in the chimeric mouse IgG2a form) in the presence of human CXCL12. Case block migration of a murine metastatic melanoma cell line (B16F10).

Figure 4B Migration traces showing the results of experiments to determine the effectiveness of the antibodies (113_1H12 (hAB113) in the human IgG2 form, 113_1H12 (mAB113) in the chimeric mouse IgG2a form, and 114_3H1 (mAB114) in the chimeric mouse IgG2a form) in blocking Migration of a Human Ovarian Cancer Cell Line (TOV-21) in the Presence of Human CXCL12.

Figure 5 Results of cell migration assays based on an in vivo experimental metastasis model of B16F10 melanoma cells CXCR4 is required for migration to the lung and initiation of metastasis.

B16F10 melanoma cells were introduced into C57B1 mice by tail vein injection on day 0, and treatment was initiated on day 1. The treatment regimen was 5 mg/kg of the clinical CXCR4 inhibitor AMD3100 (Plerixafor) twice daily or twice weekly with 10, 15 or 20 mg/kg of the anti-CXCL12 antibody mAb 114 in the chimeric murine IgG2a form -3H1. Mice in the control group were treated twice weekly with 20 mg/kg of the control antibody. All mice were sacrificed on day 14 and the number of metastatic colonies in the lungs was quantified. A level of inhibition comparable to AMD3100 was achieved with 113_1H12 at a dose of 20 mg/kg.

Figure 6 shows the results of an in vitro cell transwell migration assay in which anti-CXCL12 antibodies block the migration of TOV21G cancer cells induced by human CXCL12 in human IgG2 form.